1. Field of the Invention
This invention relates to a nucleic acid encoding a functional module or domain of a particular peptidoglycan hydrolase, i.e., the phage 2638A endolysin, a protein which specifically attacks the peptidoglycan cell wall of untreated Staphylococcus aureus and selected coagulase negative staphylococci (for example: S. chromogenes, S. simulans, S. epidermidis). The invention relates in particular to a full length construct comprising the mutant 180 codon (2638A 1-180 Mut-486) and to truncated constructs encoding a full length amidase domain and the full length SH3b domain, 2638A 139-486 and 2638A 180-486) and to the functional proteins encoded by the constructs.
2. Description of the Relevant Art
The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Staphylococcus aureus has a high negative impact worldwide as a human pathogen and also as a mastitis-causing organism based on its role in infections of dairy cattle mammary glands. S. aureus has a high capacity for resistance development. Resistant S. aureus strains exist to virtually every known antibiotic. Bacteriophage endolysins are proteins encoded by bacteriophage (viruses that infect bacteria) that help nascent phage escape their host by degrading peptidoglycan, the major structural component of bacterial cell walls. Thus, phage and host have co-evolved such that, for those species examined, no endolysin-resistant host strains have been identified (Fischetti, V. A. 2005. Trends Microbiol. 13:491-496), making phage endolysins candidate antimicrobials that are highly refractory to resistance development. To further ensure that our antimicrobials are refractory to resistance development, we have previously created fusion antimicrobials with three active lytic domains, based on the belief that no bacterium can evade three simultaneous, unique, synergistic activities (Donovan et al. 2009. Biotech International 21:6-10).
The bacterial peptidoglycans have a complex structure (sugar backbone of alternating units of N-acetyl glucosamine and N-acetyl muramic acid (NAM) residues, cross-linked by oligopeptide attachments at the NAMs). Endolysins have evolved a modular design to deal with this complexity. One protein can harbor multiple domains, each with a different peptidoglycan digestion activity. Three classes of endolysin domains have been identified thus far: the endopeptidase, glycosidase, and amidase domains (Loessner, M. J. 2005. Curr. Opin. Microbiol. 8: 480-487). Each catalytic domain has been localized to short protein domains (˜100-200 amino acids). Any one of these domains is sufficient to lyse the bacterial target cell.
It has been reported that antibiotic treatment of mastitis is less than 50% effective (Deluyker et al. 2005. J. Vet. Pharmacol. Ther. 22:274-282). S. aureus is also a notorious human pathogen with multi-drug resistant strains plaguing clinics world wide. A new antimicrobial to combat this pathogen would be an excellent addition to the collection of current treatments. There are numerous other bacteriophage endolysins that have been reported to be active against live S. aureus; for example: the phage K endolysin (O'Flaherty et al. 2005. J. Bacteriol. 187:7161-7164; Becker et al. 2008. FEMS Microbiol. Lett. 287:185-191; Becker et al. 2009a. Gene 443:32-41), the lys16 endolysin from the S. aureus phage P68 (Takac et al. 2005. Microbiol. 151:2331-2342), and the lysWMY endolysin from the Staphylococcus warneri M phage (Yokoi et al. 2005. Gene 351:97-108), to name a few.
Antibiotic resistance among pathogens is believed to develop, in part, through the use of broad range antibiotics, which affect not only the target pathogen, but can also select for resistance in other bacteria (e.g. commensals). The use of a highly specific antimicrobial would target fewer species, and thus is less likely to contribute to the broad range resistance development now apparent with commonly used broad range antibiotics. Bacteriophage endolysins are uniquely specific to their host (or closely related species); bacteriophage and bacterial hosts have co-evolved. It is difficult to prove that resistance cannot develop to endolysins, but to date, none has been reported and this fact alone makes this product a candidate for addition to the battery of antimicrobials available to both veterinary medicine and the clinician. If resistant strains are not produced, this would be an important antimicrobial for use and efficacy.
Thus, to manage the upsurge of drug resistant pathogenic bacteria, there is a need for new specific antimicrobial treatments. Reagents developed specifically for the relevant genera, species or substrains of concern would function as effective tools for controlling economically important diseases and therefore are ideal candidates for therapeutic treatments.